Terminally sterilized osteogenic devices and preparation thereof

ABSTRACT

Disclosed are terminally sterilized osteogenic devices for implantation into a mammal. The devices contain a combination of a biologically active osteogenic protein and an insoluble carrier which after being combined are sterilized by exposure to ionizing radiation, for example, by exposure to gamma rays or an electron beam. The terminally sterilized devices of the invention are characterized in that they induce bone formation following implantation into a mammal. Also disclosed is a method for inducing bone formation in a mammal by implanting a terminally sterilized device of the invention into a preselected locus in a mammal. Also disclosed is a method for preparing the terminally sterilized device of the invention.

This application is a division of Ser. No. 08/478,452, filed Jun. 7,1995 now U.S. Pat. No. 5,674,292.

TECHNICAL FIELD OF THE INVENTION

This invention relates generally to the field of osteogenic devices and,more particularly, to terminally sterilized osteogenic devices which arecapable of inducing bone formation following implantation into a mammal.

BACKGROUND OF THE INVENTION

Therapeutic devices, and more specifically, osteogenic devices,typically are sterilized prior to implantation in an intended recipient.Sterilization is required to ensure that the devices do not introducepotential pathogens, or other biologically infectious agents into theintended recipient. Osteogenic devices comprising an osteogenic proteinin combination with an insoluble carrier material are useful forinducing bone formation at a preselected locus, e.g., at a site of abone fracture, in a mammal. Heretofore, the carrier material andosteogenic protein typically have been sterilized separately and thencombined to produce a sterile implantable device. This method, however,cannot guarantee the sterility of the resulting device.

The most desirable method for sterilizing a device comprising two ormore components is by a process, referred to in the art as "terminalsterilization". By this process, the device is sterilized followingformulation, i.e., after all the components have been combined with oneanother in the device. A variety of physical or chemical methods havebeen developed for use in terminal sterilization and include, forexample, exposure to chemicals or heat, or exposure to ionizing ornon-ionizing radiation. These methods, however, can have inherentproblems.

For example, chemical reagents useful in chemical sterilization, or thereaction byproducts, can be harmful to the intended recipient.Accordingly, such chemicals must be removed prior to implantation of thedevices. Ethylene oxide and formaldehyde are reagents commonly used assterilization reagents. However, both are alkylating agents andtherefore can modify and inactivate biologically active molecules. Inaddition, both of these chemicals are carcinogens and mutagens (Davis etal., (1973) "Microbiology, 2nd Ed.", Harper and Row, Publishers).Similarly, where the device requires a biologically active protein,exposing the device to elevated temperatures is not desirable becausethe proteins can be denatured and subsequently inactivated by exposureto heat. Although the sterilization of objects by exposure to ionizingand non-ionizing radiation obviates the necessity of adding potentiallytoxic chemicals, the radiation energy and/or its byproducts, includingoxygen free radicals, are competent to modify protein conformation andso can damage or inactivate the protein, In addition, exposure of somemedically important polymers, for example, as polyurethane orpolymethylmethacrylate to gamma radiation can result in immediate andlong term physical changes to the polymer.

It is therefore an object of this invention to provide a terminallysterilized osteogenic device which, when implanted at a preselectedlocus in a mammal, is capable of producing bone at the locus. Anotherobject is to provide a general process for terminally sterilizingosteogenic devices without compromising the biological activity and/orbiocompatibility of the device. Another object of the invention is toprovide a method of inducing bone formation at a preselected locus in amammal using a terminally sterilized device of the invention.

These and other objects and features of the invention will be apparentfrom the description, drawings, and claims which follow.

SUMMARY OF THE INVENTION

It now has been discovered that a terminally sterilized therapeuticdevice, specifically an osteogenic device, comprising a biologicallyactive protein, for example, an osteogenic protein, in combination withan insoluble carrier material, when sterilized by exposure to ionizingradiation is capable of inducing bone and/or cartilage formation whenimplanted into a mammal. The finding is unexpected as it is known thatexposure of biologically active proteins to ionizing radiation canresult in chemical modification and inactivation of the protein.

In its broadest aspect, the invention provides a terminally sterilizedosteogenic device for implantation into a mammal which, when implantedinto the mammal, induces bone and/or cartilage formation. The device isproduced by the steps of (a) combining an insoluble carrier and abiologically active osteogenic protein to form an osteogenic device, andthen (b) exposing the combination of step (a) to ionizing radiationunder conditions to sterilize the device while maintaining biologicalactivity of the osteogenic protein. The resulting sterile device ischaracterized in that it has been terminally sterilized but yet iscapable of inducing bone and/or cartilage formation followingimplantation into the mammal.

The term, "sterilization" as used herein, refers to an act or processusing either physical or chemical means for eliminating substantiallyall viable organisms, especially micro-organisms, viruses and otherpathogens, associated with an osteogenic device. As used herein,sterilized devices are intended to include devices achieving a sterilityassurance level of 10⁻⁶, as determined by FDA (Federal DrugAdministration) standards. The term, "terminal sterilization" as usedherein, refers to the last step in the fabrication of the device of theinvention wherein the insoluble carrier material is sterilized afterbeing combined with the osteogenic protein. The term "ionizingradiation" as used herein, refers to particles or photons that havesufficient energy to produce ionization directly in their passagethrough a substance, e.g., the therapeutic device contemplated herein.

The term, "osteogenic device" as used herein, refers to any devicehaving the ability, when implanted into a mammal, to induce boneformation. The device described herein also is competent to inducearticular cartilage formation when implanted at an avascular site in amammal, such as at the surface of subchondral bone in a synovial jointenvironment. As used herein, the term "bone" refers to a calcified(mineralized) connective tissue primarily comprising a composite ofdeposited calcium and phosphate in the form of hydroxyapatite collagen(predominantly Type I collagen) and bone cells, such as osteoblasts,osteocytes and osteoclasts, as well as to the bone marrow tissue whichforms in the interior of true endochondral bone.

As used herein, the term "cartilage" refers to a type of connectivetissue that contains chondrocytes embedded in an extracellular networkcomprising fibrils of collagen (predominantly Type II collagen alongwith other minor types, e.g. Types IX and XI), various proteoglycans(e.g., chondroitin sulfate, keratan sulfate, and dermatan sulfateproteoglycans), other proteins, and water. "Articular cartilage" refersto hyaline or articular cartilage, an avascular, non-mineralized tissuewhich covers the articulating surfaces of bones in joints and allowsmovement in joints without direct bone-to-bone contact, and therebyprevents wearing down and damage to opposing bone surfaces. Most normalhealthy articular cartilage is referred to as "hyaline," i.e., having acharacteristic frosted glass appearance. Under physiological conditions,articular cartilage tissue rests on the underlying mineralized bonesurface, the subchondral bone, which contains highly vascularizedossicles. These highly vascularized ossicles can provide diffusiblenutrients to the overlying cartilage, but not mesenchymal stem cells.

As used herein, the term "osteogenic protein" is understood to mean anyprotein capable of producing, when implanted into a mammal, adevelopmental cascade of cellular events resulting in endochondral boneformation. The developmental cascade occurring during endochondral bonedifferentiation consists of chemotaxis of mesenchymal cells,proliferation of progenitor cells into chondrocytes and osteoblasts,differentiation of cartilage, vascular invasion, bone formation,remodeling, and finally marrow differentiation. True osteogenic factorscapable of inducing the above-described cascade of events that result inendochondral bone formation have now been identified, isolated, andcloned. These proteins, which occur in nature as disulfide-bondeddimeric proteins, are referred to in the art as "osteogenic" proteins,"osteoinductive" proteins, and "bone morphogenetic" proteins. Osteogenicprotein can be, for example, any of the known bone morphogeneticproteins and/or equivalents thereof described herein and/or in the artand includes naturally sourced material, recombinant material, and anymaterial otherwise produced which is capable of inducing tissuemorphogenesis. Osteogenic protein as defined herein also is competent toinduce articular cartilage formation at an appropriate in vivo avascularlocus.

As used herein, the term "carrier material" is understood to mean amaterial having interstices for the attachment, proliferation, anddifferentiation of infiltrating cells. It is biodegradable in vivo andit is biocompatible. That is, it is sufficiently free of antigenicstimuli which can result in graft rejection. Preferably, the carriercomprises insoluble material and further is formulated to have a shapeand dimension when implanted which substantially mimics that of thereplacement bone or cartilage tissue desired. The carrier further cancomprise residues specific for the tissue to be replaced and/or derivedfrom the same tissue type.

In a preferred embodiment, the weight ratio of osteogenic protein tocarrier material preferably is within the range from about 1:1 to about1:250,000 (e.g., from about 1 mg protein: 1 mg carrier to about 4 ngprotein: 1 mg carrier) and most preferably in the range from about 1:40to about 1:50,000 (e.g., from about 25 μg protein: 1 mg of carrier toabout 20 ng protein: 1 mg carrier).

In one embodiment, the ionizing radiation is an electron beam. Inanother embodiment, gamma radiation is the preferred source of ionizingradiation. It is contemplated that any conventional gamma ray orelectron beam-producing device may be used in the practice of theinvention. Furthermore, the preferred dosage of ionizing radiation isprovided within the range of about 0.5 to about 4.0 megarads, and mostpreferably within the range of about 2.0 to about 3.5 megarads, whichare doses sufficient to produce the FDA required sterility assurancelevel of 10⁻⁶ for the devices described herein. The dosages required forobtaining a sterility assurance level of 10⁻⁶ for a particular device,however, can be determined from the "Association for the Advancement ofMedical Instrumentation Guidelines" published in 1992, the disclosure ofwhich is incorporated herein by reference.

In another embodiment, the insoluble carrier material comprises porousmaterial which further can be particulate. The pores preferably havedimensions that are sufficient to permit the entry and subsequentdifferentiation and proliferation of migratory progenitor cells in thematrices. Alternatively, the insoluble carrier material can befabricated by closely packing the particulate material into a shapesuitable for an intended use in vivo, for example, in spanning bonedefects. The porous particles or packed particles preferably have aparticle size within the range of about 70 to about 850 microns and mostpreferably within the range of about 125 to about 450 microns. Inanother embodiment, the carrier material is formulated as part of anarticular cartilage device. The device can be formed from devitalizedcartilage tissue, or other inert, non-mineralized matrix material andosteogenic protein, and the device laid on the subchondral bone surfaceas a sheet. Alternatively, a formulated device can be pulverized orotherwise mechanically abraded to produce particles which can beformulated into a paste or gel as described herein for application tothe bone surface.

The insoluble carrier material can comprise a non-protein-based polymer,for example, a synthetic polymer comprising polylactic acid, polybutyricacid, polyglycolic acid, and/or mixtures thereof; and/or one or morenaturally derived molecules, for example, hydroxyapatite, tricalciumphosphate, collagen and mixtures thereof. Collagen currently is apreferred carrier material. A person of ordinary level of skill in theart, by judicious choice of natural and/or synthetic materials, cangenerate polymeric matrices that have the desired in vivo physical andchemical properties. For example, autologous collagen can be mixed withsynthetic polymers, including copolymers, to produce a matrix having anenhanced in vivo biodegradation rate, and/or to improve the preferredhandling qualities which make the device of the invention more easy tomanipulate during implantation. For example, particulatecollagen-containing devices can be combined with one or more componentswhich serve to bind the particles into a paste-like or gel-likesubstance. Binding materials well characterized in the art include, forexample, carboxymethylcellulose, glycerol, polyethylene-glycol and thelike. Alternatively, the device can comprise osteogenic proteindispersed in a synthetic matrix which provides the desired physicalproperties.

The osteogenic protein useful in the methods and devices of theinvention, whether naturally-occurring or synthetically prepared, iscapable of inducing recruitment of accessible progenitor cells andstimulating their proliferation, inducing differentiation intochondrocytes and osteoblasts, and further inducing differentiation ofintermediate cartilage, vascularization, bone formation, remodeling, andfinally marrow differentiation when implanted in a mammal. The proteinalso is competent to induce new articular cartilage tissue formation ona subchondral bone surface, when provided in an appropriate localenvironment.

Preferred osteogenic proteins include, for example, homo- orheterodimers of OP-1, OP-2, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 orfunctional equivalents thereof. These proteins are referred to in theart as members of the "Vg/dpp" protein subfamily of the TGF-β super genefamily.

In another aspect, the invention provides a method for inducing boneformation and/or articular cartilage formation in a mammal. The methodcomprises the steps of (a) implanting at a pre-selected locus in themammal a terminally sterilized device of the invention and (b)permitting the device to induce the appropriate tissue formation at thepreselected locus.

In another aspect, the invention provides a general procedure forproducing a terminally sterilized osteogenic device suitable forimplantation into a mammal. The method comprises the steps of (a)providing a biologically active osteogenic protein; (b) combining theosteogenic protein with an insoluble carrier material; and (c) exposingthe combination to ionizing radiation in an amount sufficient toterminally sterilize the combination while maintaining biologicalactivity of the protein. The process is rapid, gentle and is performedusing conventional irradiation devices. The invention contemplates boththe sterilization process and the sterilized products produced by themethod.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of the invention, the various featuresthereof, as well as the invention itself, may be more fully understoodfrom the following description, when read together with the accompanyingdrawing, in which:

FIG. 1 is a graph depicting the ability of samples extracted fromirradiated and non-irradiated osteogenic devices to stimulate alkalinephosphatase activity in a rat osteoblast cell assay. The trace havingthe filled triangles depicts a sample extracted from control matrix notcontaining OP-1; the trace having filled squares depicts a sample ofuntreated OP-1; the trace having crosses depicts a sample extracted froma first non-irradiated OP-1 containing device; the trace havingasterisks depicts a sample extracted from the first irradiated OP-1containing device; the trace having the unfilled squares depicts asample extracted from a second non-irradiated OP-1 containing device;and the trace having the rotated crosses depicts a sample extracted fromthe second irradiated OP-1 containing device.

DETAILED DESCRIPTION OF THE INVENTION

It now has been discovered that an osteogenic device, terminallysterilized by exposure to ionizing radiation and comprising anosteogenic protein in combination with a carrier material, retains itsbiological activity after sterilization and is competent to induceendochondral bone formation and/or cartilage formation when implantedinto a mammal. The discovery is unexpected as ionizing radiation canmodify protein structure and thereby destroying biological activity.

The general procedure described herein ensures sterility of theosteogenic device while maintaining the biological activity of theosteogenic protein incorporated in the device. The procedure involvesthe steps of combining an insoluble carrier material and a biologicallyactive osteogenic protein and then terminally sterilizing thecombination by exposure to ionizing radiation, thereby to produce asterile device which induces bone formation following implantation intothe mammal. The method may be used for a variety of osteogenic proteins,carrier matrices and formulations thereof. The method also can be usedto create devices competent to induce articular cartilage formation atan avascular site in vivo.

The preparation of terminally sterilized osteogenic devices having boneand articular cartilage forming activity in vivo, suitable osteogenicproteins, the nature and properties of the carrier material, treatmentsto minimize protein modification, conditions which enable terminalsterilization, and other material aspects concerning the nature andutility of the invention, including how to make and how to use thesubject matter claimed herein will be further understood from thefollowing.

I. Osteogenic Proteins

As defined herein, the osteogenic proteins useful in the composition andmethods of the invention include the family of dimeric proteins havingendochondral bone activity when implanted in a mammal in associationwith a matrix and which comprise a subclass of the "super family" of"TGF-β-like" proteins. Natural-sourced osteogenic protein in its mature,native form is a glycosylated dimer typically having an apparentmolecular weight of about 30-36 kDa as determined by SDS-PAGE. Whenreduced, the 30 kDa protein gives rise to two glycosylated peptidesubunits having apparent molecular weights of about 16 kDa and 18 kDa.In the reduced state, the protein has no detectable osteogenic activity.The unglycosylated protein dimer, which also has osteogenic activity,has an apparent molecular weight of about 27 kDa. When reduced, the 27kDa protein gives rise to two unglycosylated polypeptides havingmolecular weights of about 14 kDa to 16 kDa. Useful sequences includethose comprising the C-terminal 102 amino acid sequences of DPP (fromDrosophila), Vg1 (from Xenopus), Vgr-1 (from mouse), the OP-1 and OP-2proteins (see U.S. Pat. No. 5,011,691), as well as the proteins referredto as BMP2, BMP3, BMP4 (see W088/00205, U.S. Pat. No. 5,013,649 andW091/18098), BMP5 and BMP6 (see WO90/11366, PCT/US90/01630), and BMP8and 9.

The members of this family of proteins share a conserved six or sevencysteine skeleton in the C-terminal region. See, for example, residues335-431 of Seq. ID No. 1 in U.S. Pat. No. 5,266,683, the disclosure ofwhich is incorporated herein by reference, which defines the sixcysteine skeleton residues referred to herein as "OPS", or residues330-431 of Seq. ID No. 1 therein, comprising 102 amino acids whichdefines the seven cysteine skeleton.

This family of proteins includes longer forms of a given protein, aswell as phylogenetic forms, e.g., species and allelic variants andbiosynthetic mutants, including addition and deletion mutants andvariants such as those which may alter the conserved C-terminal cysteineskeleton, provided that the alteration still allows the protein to forma dimeric species having a conformation capable of inducing boneformation in a mammal when implanted in the mammal in association with amatrix. In addition, the osteogenic proteins useful in devices of thisinvention may include forms having varying glycosylation patterns andvarying N-termini. The osteogenic proteins may be naturally occurring orbiosynthetically derived, and may be produced by expression ofrecombinant DNA in prokaryotic or eukaryotic host cells. The proteinsare active as a single species, e.g., a homodimer, or combined as amixed species,.e.g., a heterodimer.

In one embodiment, the osteogenic protein contemplated herein comprisesOP-1 or an OP-1-related sequence. Useful OP-1 sequences are recited inU.S. Pat. Nos. 5,011,691; 5,018,753 and 5,266,683; in Ozkaynak et al.(1990) EMBO J. 9: 2085-2093; and Sampath et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90: 6004-6008, the disclosures of which are incorporatedherein by reference. OP-1 related sequences include xenogenic homologs,e.g.; 60A, from Drosophila, (Wharton et al. (1991) Proc. Natl. Acad.Sci. U.S.A. 88: 9214-9218) and proteins sharing greater than 60%identity with OP-1 in the C-terminal seven cysteine domain, preferablyat least 65% identity. Examples of OP-1 related sequences include OP-2,BMP5, BMP6 and its species homolog vgr-1 (Lyons et al. (1989) Proc.Natl. Acad. Sci. U.S.A. 86: 4554-4558; Celeste, et al. (1990) Proc Natl.Acad. Sci. U.S.A. 87: 9843-9847; and PCT international applicationWO93/00432; Ozkaynak et al. (1992) J. Biol. Chem. 267: 13198-13205). ASwill be appreciated by those having ordinary skill in the art, chimericconstructs can be created using standard molecular biology andmutagenesis techniques combining various portions of differentmorphogenic protein sequences to create a novel sequence, and theseforms of the protein also are contemplated herein.

In still another preferred aspect, one or both of the polypeptide chainsubunits of the osteogenically active dimer is encoded by nucleic acidswhich hybridize to DNA or RNA sequences encoding the active region ofOP-1 under stringent hybridization conditions. As used herein, stringenthybridization conditions are defined as hybridization in 40% formamide,5×SSPE, 5×Denhardt's Solution, and 0.1% SDS at 37° C. overnight, andwashing in 0.1×SSPE, 0.1% SDS at 50° C.

Given the foregoing amino acid and DNA sequence information, the levelof skill in the art, and the disclosures of numerous publications onosteogenic proteins, including U.S. Pat. No. 5,011,691 and published PCTspecification U.S. 89/01469 (published Oct. 19, 1989) various DNAs canbe constructed which encode at least the active domain of an osteogenicprotein useful in the devices of this invention, and various analogsthereof (including species and allelic variants and those containinggenetically engineered mutations), as well as fusion proteins, truncatedforms of the mature proteins, deletion and addition mutants, and similarconstructs which can be used in the devices and methods of theinvention. Moreover, DNA hybridization probes can be derived fromfragments of any of these proteins, or designed de novo from the genericsequence defined above as OPS. These probes then can be used to screendifferent genomic and cDNA libraries to identify additional osteogenicproteins useful in the prosthetic devices of this invention.

The DNAs can be produced by those skilled in the art using well knownDNA manipulation techniques involving genomic and cDNA isolation,construction of synthetic DNA from synthesized oligonucleotides, andcassette mutagenesis techniques. 15-100 mer oligonucleotides may besynthesized on a DNA synthesizer, and purified by polyacrylamide gelelectrophoresis (PAGE) in Tris-Borate-EDTA buffer. The DNA then may beelectroeluted from the gel. Overlapping oligomers may be phosphorylatedby T4 polynucleotide kinase and ligated into larger blocks which mayalso be purified by PAGE.

The DNA from appropriately identified clones then can be isolated,subcloned (preferably into an expression vector), and sequenced.Plasmids containing sequences of interest then can be transfected intoan appropriate host cell for protein expression and furthercharacterization. The host may be a prokaryotic or eucaryotic cell sincethe former's inability to glycosylate protein will not destroy theprotein's morphogenic activity. Useful host cells include E. coli,Saccharomyces, the insect/baculovirus cell system, myeloma cells, CHOcells and various other mammalian cells. The vectors additionally mayencode various sequences to promote correct expression of therecombinant protein, including transcription promoter and terminationsequences, enhancer sequences, preferred ribosome binding sitesequences, preferred MRNA leader sequences, preferred signal sequencesfor protein secretion, and the like.

The DNA sequence encoding the gene of interest also may be manipulatedto remove potentially inhibiting sequences or to minimize unwantedsecondary structure formation. The recombinant osteogenic protein alsomay be expressed as a fusion protein. After being translated, theprotein may be purified from the cells themselves or recovered from theculture medium. All biologically active protein forms comprise dimericspecies joined by disulfide bonds or otherwise associated, produced byfolding and oxidizing one or more of the various recombinant polypeptidechains within an appropriate eucaryotic cell or in vitro afterexpression of individual subunits. A detailed description of osteogenicproteins expressed from recombinant DNA in E. coli and in numerousdifferent mammalian cells is disclosed in U.S. Pat. No. 5,266,683.

Alternatively, osteogenic polypeptide chains can be synthesizedchemically using conventional peptide synthesis techniques well known tothose having ordinary skill in the art. For example, the proteins may besynthesized intact or in parts on a solid phase peptide synthesizer,using standard operating procedures. Completed chains then aredeprotected and purified by HPLC (high pressure liquid chromatography).If the protein is synthesized in parts, the parts may be peptide bondedusing standard methodologies to form the intact protein. In general, themanner in which the osteogenic proteins are made is conventional anddoes not form a part of this invention.

II. Carrier Matrix Material

A. General Matrix Considerations

As will be appreciated by the skilled artisan, provided the matrix has athree dimensional structure sufficient to act as a scaffold forinfiltrating and proliferating cells, and is bioresorbable biocompatiblein vivo, the precise nature of the substrate per se used for thematrices disclosed herein is not determinative of a device's ultimateability to induce new bone or articular cartilage tissue formation. Inthe instant invention, the substrate serves as a scaffold upon whichcertain cellular events, mediated by an osteogenic protein, may occur.The specific responses to the osteogenic protein ultimately are dictatedby the endogenous microenvironment at the implant site and thedevelopmental potential of the responding cells. As also will beappreciated by the skilled artisan, the precise choice of substrateutilized for the matrices disclosed herein will depend, in part, uponthe type of defect to be repaired, anatomical considerations such as theextent of vascularization at the defect site, and the like.

Matrix geometry, particle size, the presence of surface charge, and thedegree of porosity (cell infiltrating interstices) all are important tosuccessful matrix performance. It is preferred to shape the matrix tothe desired form of the new bone or articular cartilage tissue to beformed. Rat studies show that the new bone is formed essentially havingthe dimensions of the device implanted.

The matrix can comprise a shape-retaining solid made of loosely adheredparticulate material, e.g., with collagen. It also can comprise amolded, porous solid, or simply an aggregation of close-packed particlesheld in place by surrounding tissue. The matrix further can comprise aninsoluble, non-particulate solid with interstices sufficient to allowthe attachment and proliferation of infiltrating cells. Masticatedmuscle or devitalized, biologically inert tissue can be used,particularly when prepared as described herein. Large allogenic boneimplants also can act as a carrier for the matrix if their marrowcavities are cleaned and packed with carrier and the dispersedosteogenic protein. Alternatively, the bone implants may act as acarrier per se and in such cases the osteogenic protein may be coateddirectly onto the surface of the bone implant.

Where the osteogenic device is formulated to form new bone tissue in amammal, the currently preferred matrix material comprises devitalized,demineralized xenogenic bone tissue, treated as disclosed herein. Theformulated devices produce an implantable material useful in a varietyof clinical settings. In addition to its use as a matrix for boneformation in various orthopedic, periodontal, and reconstructiveprocedures, the matrix also can be used as a sustained release carrier,or as a collagenous coating for implants. The matrix can be shaped asdesired in anticipation of surgery or shaped by the physician ortechnician during surgery. Thus, the material can be used for topical,subcutaneous, intraperitoneal, or intramuscular implants; it can beshaped to span a nonunion fracture or to fill a bone defect.

B. Natural-sourced Matrices

Suitable allogenic or xenogenic matrices can be created as describedherein below, using methods well known in the art. Specifically, themethods are designed to extract the cellular, non-structural componentsof the tissue so as to devitalize the tissue. The resulting materialcomprises an cellular matrix defining interstices that can beinfiltrated by cells and which is substantially depleted innon-structurally associated components.

A currently preferred procedure for devitalizing nonmineralized tissuefollows a methodology such as that used in the art for fixing tissue.The tissue is exposed to a non-polar solvent, such as 95% ethanol, for atime sufficient to substantially replace the water content of the tissuewith ethanol and to destroy the cellular structure of the tissue.Typically, the tissue is exposed to 200 proof ethanol for several days,at a temperature in the range of about 4°-40° C., taking care to replacethe solution with fresh ethanol every 6-12 hours, until such time as theliquid content of the tissue comprises 70-90% ethanol. Typically,treatment for 3-4 days is appropriate. The volume of liquid added shouldbe more than enough to submerge the tissue. The treated tissue then islyophilized. The resulting, dry matrix is substantially depleted innon-structural components but retains both intracellular andextracellular matrix components derived from the tissue.

Treated allogenic or xenogenic matrices are envisioned to haveparticular utility for creating devices for forming new bone orarticular cartilage in a mammal. An osteogenic device can be formulatedfrom an allogenic bone to enhance allograft repair. Devitalizedallogenic or xenogenic carrier material also can be combined withosteogenic protein to provide a solid, resorbable matrix which providesstructural support for large bony defects. Similarly, an allogenicarticular cartilage device can be formed from devitalized cartilagetissue, or other inert non-mineralized matrix material and osteogenicprotein, and the device laid on the subchondral bone surface as a sheet.Alternatively, a formulated device can be pulverized or otherwisemechanically abraded to produce particles which can be formulated into apaste or gel as described herein, for application to the bone surface.

C. Bone-Derived Matrices

The following provide currently preferred methodologies for creatingappropriate matrices from mineralized tissue, particularly allogenic orxenogenic bone tissue.

C.1 Demineralized Bone Matrix

Demineralized bone matrix, preferably bovine bone matrix, is prepared bypreviously published procedures (Sampath et al. (1983) Proc. Natl. Acad.Sci. U.S.A. 80: 6591-6595). Bovine diaphyseal bones are obtained from alocal slaughterhouse and used fresh. The bones are stripped of muscleand fat, cleaned of periosteum, demarrowed by pressure with cold water,dipped in cold absolute ethanol, and stored at -20° C. Then, they aredried and fragmented by crushing and pulverized in a large mill. Care istaken to prevent heating by using liquid nitrogen. The pulverized boneis milled to a particle size in the range of 70-850 μm, preferably150-420 μm, and is defatted by two washes of approximately two hoursduration and three volumes of chloroform: methanol (3:1). Theparticulate bone then is washed with one volume of absolute ethanol anddried over one volume of anhydrous ether yielding defatted bone powder.The defatted bone powder then is demineralized by four successivetreatments with 10 volumes of 0.5N HCl at 4° C. for 40 min. Finally, thedemineralized bone powder is neutralized by washing with a large volumeof water.

C. 2. Guanidine Extraction

Demineralized bone matrix thus prepared is extracted with 5 volumes of4M guanidine-HCl, 50 mM Tris-HCl, pH 7.0 for 16 hr at 4° C. Thesuspension is filtered. The insoluble material is collected and used tofabricate the matrix. The resulting material is mostly collagenous innature and is devoid of osteogenic or chondrogenic activity.

C. 3. Matrix Treatments

The major component of all bone matrices is Type-I collagen. In additionto collagen, demineralized bone extracted as disclosed above includesnon-collagenous proteins which may account for 5% of its mass. In axenogenic matrix, these noncollageneous components may presentthemselves as potent antigens, and may constitute immunogenic and/orinhibitory components. These components also may inhibit osteogenesis inallogenic implants by interfering with the developmental cascade of bonedifferentiation. It has been discovered that treatment of the matrixparticles with a collagen fibril-modifying agent extracts potentiallyunwanted components from the matrix, and alters the surface structure ofthe matrix material. Useful agents include acids, organic solvents orheated aqueous media. Various treatments are described below. A detailedphysical analysis of the effect that these fibril-modifying agents haveon demineralized, quanidine-extracted bone collagen particles isdisclosed in copending U.S. patent application Ser. No. 483,913, filedFeb. 22, 1990.

After contact with the fibril-modifying agent, the treated matrix iswashed to remove any extracted components. Briefly, the matrix issuspended in TBS (Tris-buffered saline) 1 g/200 ml and stirred at 4° C.for 2 hrs; or in 6M urea, 50 mM Tris-HCl, 500 mM NaCl, pH 7.0 (UTBS)stirred at room temperature (RT) for 30 minutes (sufficient time toneutralize the pH). The material is harvested by centrifugation,rewashed using the aforementioned conditions, and reharvested bycentrifugation. The resulting material is washed with water and thenlyophilized.

C. 3.1. Acid Treatments

C. 3.1a. Trifluoroacetic acid

Trifluoroacetic acid (TFA) is a strong non-oxidizing acid that is aknown swelling agent for proteins, and which modifies collagen fibrils.Bovine bone residue prepared as described above is sieved, and particlesof the appropriate size are collected. These particles are extractedwith various percentages (1.0% to 100%) of trifluoroacetic acid andwater (v/v) at 0° C. or room temperature for 1-2 hours with constantstirring. The treated matrix is filtered, lyophilized, or washed withwater/salt and then lyophilized.

C. 3.1b. Hydrogen Fluoride

Like trifluoroacetic acid, hydrogen fluoride (HF) is a strong acid andswelling agent, and also is capable of altering intraparticle surfacestructure. Hydrogen fluoride also is a known deglycosylating agent. Assuch, hydrogen fluoride may function to increase the osteogenic activityof these matrices by removing the antigenic carbohydrate content of anyglycoproteins still associated with the matrix after guanidineextraction.

Bovine bone residue prepared as described above is sieved, and particlesof the appropriate size are collected. The sample is dried in vacuo overP₂ O₅, transferred to the reaction vessel and exposed to anhydroushydrogen fluoride (10-20 ml/g of matrix) by distillation onto the sampleat -70° C. The vessel is allowed to warm to 0° C. and then the reactionmixture is stirred at this temperature for 120 minutes. Afterevaporation of the hydrogen fluoride in vacuo, the residue is driedthoroughly over KOH pellets in vacuo to remove any remaining traces ofacid. Extent of deglycosylation can be determined from carbohydrateanalysis of matrix samples taken before and after treatment withhydrogen fluoride, after washing the samples appropriately to removenon-covalently bound carbohydrate. SDS-extracted protein from HF-treatedmaterial is negative for carbohydrate as determined by Con A blotting.Then, the deglycosylated bone matrix is washed twice in TBS(Tris-buffered saline) or UTBS, then washed with water and thenlyophilized. Other acid treatments, however, are envisioned in additionto HF and TFA.

C. 3.2 Solvent Treatment

C. 3.2a. Dichloromethane

Dichloromethane (DCM) is an organic solvent capable of denaturingproteins without affecting their primary structure. This swelling agentis a common reagent in automated peptide synthesis, and is used inwashing steps to remove components. Bovine bone residue, prepared asdescribed above, is sieved, and particles of the appropriate size areincubated in 100% DCM or, preferably, 99.9% DCM/0.1% TFA. The matrix isincubated with the swelling agent for one or two hours at 0° C. or atroom temperature. Alternatively, the matrix is treated with the agent atleast three times with short washes (20 minutes each) with noincubation.

C. 3.2b. Acetonitrile

Acetonitrile (ACN) is an organic solvent, capable of denaturing proteinswithout affecting their primary structure. It is a common reagent usedin a high-performance liquid chromatography, and is used to eluteproteins from silica-based columns by perturbing hydrophobicinteractions. Bovine bone residue particles of the appropriate size,prepared as described above, are treated with 100% ACN (1.0 g/30 ml) or,preferably, 99.9% ACN/0.1% TFA at room temperature for 1-2 hours withconstant stirring. The treated matrix is then washed with water, a ureabuffer, or 4M NaCl and then lyophilized. Alternatively, the ACN orACN/TFA treated matrix may be lyophilized without washing.

C. 3.2c. Isopropanol

Isopropanol also is an organic solvent capable of denaturing proteinswithout affecting their primary structure. It is a common reagent usedto elute proteins from silica HPLC columns. Bovine bone residueparticles of the appropriate size prepared as described above aretreated with 100% isopropanol (1.0 g/30 ml) or, preferably, in thepresence of 0.1% TFA, at room temperature for 1-2 hours with constantstirring. The matrix then is washed with water, urea buffer, or 4 M NaClbefore being lyophilized.

C. 3.2d. Chloroform

Chloroform also may be used to increase surface area of bone matrix likethe reagents set forth above, either alone or acidified. Treatment asset forth above is effective to assure that the material is free ofpathogens prior to implantation.

C. 3.3 Heat Treatment

The currently most preferred agent is a heated aqueous fibril-modifyingmedium such as water, to increase the matrix particle surface area andporosity. The currently most preferred aqueous medium is an acidicaqueous medium having a pH of less than about 4.5, e.g., within therange of about pH 2-pH 4 which may help to "swell" the collagen beforeheating. 0.1% acetic acid, which has a pH of about 3, currently is mostpreferred. 0.1 M acetic acid also may be used.

Various amounts of delipidated, demineralized guanidine-extracted bonecollagen are heated in the aqueous medium (1 g matrix/30 ml aqueousmedium) under constant stirring in a water jacketed glass flask, andmaintained at a given temperature for a predetermined period of time.Preferred treatment times are about one hour, although exposure times ofbetween about 0.5 to two hours appear acceptable. The temperatureemployed is held constant at a temperature within the range of about 37°C. to 65° C. The currently preferred heat treatment temperature iswithin the range of about 45° C. to 65° C.

After the heat treatment, the matrix is filtered, washed, lyophilizedand used for implant. Where an acidic aqueous medium is used, the matrixalso is preferably neutralized prior to washing and lypohilization. Acurrently preferred neutralization buffer is a 200 mM sodium phosphatebuffer, pH 7.0. To neutralize the matrix, the matrix preferably first isallowed to cool following thermal treatment, the acidic aqueous medium(e.g., 0.1% acetic acid) then is removed and replaced with theneutralization buffer and the matrix agitated for about 30 minutes. Theneutralization buffer then may be removed and the matrix washed andlyophilized.

The matrix also may be treated to remove contaminating heavy metals,such as by exposing the matrix to a metal ion chelator. For example,following thermal treatment with 0.1% acetic acid, the matrix may beneutralized in a neutralization buffer containing sodiumethylenediamietetraacetic acid (EDTA), e.g., 200 mM sodium phosphate, 5mM EDTA, pH 7.0. 5 mM EDTA provides about a 100-fold molar excess ofchelator to residual heavy metals present in the most contaminatedmatrix tested to date. Subsequent washing of the matrix followingneutralization appears to remove the bulk of the EDTA. EDTA treatment ofmatrix particles reduces the residual heavy metal content of all metalstested (Sb, As, Be, Cd, Cr, Cu, Co, Pb, Hg, Ni, Se, Ag, Zn, Tl) to lessthan about 1 ppm. Bioassays with EDTA-treated matrices indicate thattreatment with the metal ion chelator does not inhibit bone inducingactivity.

The collagen matrix materials preferably take the form of a fine powder,insoluble in water, comprising nonadherent particles. It may be usedsimply by packing into the volume where new bone growth or sustainedrelease is desired, held in place by surrounding tissue. Alternatively,the powder may be encapsulated in, e.g., a gelatin or polylactic acidcoating, which is absorbed readily by the body. The powder may be shapedto a volume of given dimensions and held in that shape by interadheringthe particles using, for example, soluble species-biocompatiblecollagen. The material also may be produced in sheet, rod, bead, orother macroscopic shapes.

D. Synthetic Matrices

As an alternative to a natural-sourced matrix, or as a supplement to beused in combination with a natural-sourced matrix, a suitable matrixalso can be formulated de novo, using one or more materials which serveto create a three-dimensional scaffolding structure that can be formedor molded to take on the dimensions of the replacement tissue desired.Preferably, the matrix also comprises residues derived from and/orcharacteristic of, or specific for, the same tissue type as the tissueto be induced. In some circumstances, as in the formation of articularcartilage on a subchondral bone surface, osteogenic protein incombination with a matrix defining a three-dimensional scaffoldingstructure sufficient to allow the attachment of infiltrating cells andcomposed of a non-mineralized material can be sufficient. Any one orcombination of materials can be used to advantage, including, withoutlimitation, collagen; homopolymers or copolymers of glycolic acid,lactic acid, and butyric acid, including derivatives thereof; andceramics, such as hydroxyapatite, tricalcium phosphate and other calciumphosphates and combinations thereof.

The tissue-specific component of a synthetic matrix readily can beobtained by devitalizing an allogenic or xenogenic tissue as describedabove and then pulerizing or otherwise mechanically breaking down theinsoluble matrix remaining. This particulate material then can becombined with one or more structural materials, including thosedescribed herein. Alternatively, tissue-specific components can befurther purified from the treated matrix using standard extractionprocedures well characterized in the art and, using standard analysisprocedures, the extracted material at each purification step can betested for its tissue-specificity capability. For exemplary tissueextraction protocols, see, for example, Sampath et al. (1987) Proc.Natl. Acad. Sci 78: 7599-7603 and U.S. Pat. No. 4,968,590, thedisclosures of which are incorporated herein by reference.

A synthetic matrix may be desirable where, for example, replacementarticular cartilage is desired in an existing joint to, for example,correct a tear or limited superficial defect in the tissue, or toincrease the height of the articular cartilage surface now worn due toage, disease or trauma. Such "resurfacing" of the articular cartilagelayer can be achieved using the methods and compositions of the instantinvention by, in one embodiment, treating a sheet of allogenic orxenogenic articular cartilage tissue as described herein, coating theresulting matrix with osteogenic protein, rolling up the formulateddevice so that it can be introduced to the joint using standardorthoscopic surgical techniques and, once provided to the site,unrolling the device as a layer onto the articular bone surface.

In another embodiment, the device is formulated as a paste or injectablegel-like substance that can be injected onto the articular bone surfacein a joint, using standard orthoscopic surgical techniques. In thisembodiment, the formulation may comprise a pulverized or otherwisemechanically degraded device comprising both matrix and osteogenicprotein and, in addition, one or more components which serve to bind theparticles into a paste-like or gel-like substance. Binding materialswell characterized in the art include, for example,carboxymethylcellulose, glycerol, polyethylene-glycol and the like.Alternatively, the device can comprise osteogenic protein dispersed in asynthetic matrix which provides the desired physical properties.

As an example, a synthetic matrix having tissue specificity forcartilage and bone is described in WO91/18558 (published Dec. 21, 1991).Briefly, the matrix comprises a porous crosslinked structural polymer ofbiocompatible, biodegradable collagen and appropriate, tissue-specificglycosaminoglycans as tissue-specific cell attachment factors. Collagenderived from a number of sources can be used, including insolublecollagen, acid-soluble collagen, collagen soluble in neutral or basicaqueous solutions, as well as those collagens which are commerciallyavailable.

Glycosaminoglycans (GAGs) or mucopolysaccharides arehexosamine-containing polysaccharides of animal origin that have atissue specific distribution, and therefore may be used to helpdetermine the tissue specificity of the morphogen-stimulateddifferentiating cells. Reaction with the GAGs also provides collagenwith another valuable property, i.e., inability to provoke an immunereaction (foreign body reaction) from an animal host.

Chemically, GAGs are made up of residues of hexoamines glycosidicallybound and alternating in a more-or-less regular manner with eitherhexouronic acid or hexose moieties (see, e.g., Dodgson et al. in"Carbohydrate Metabolism and its Disorders", Dickens et al., eds. vol.1, Academic Press (1968)). Useful GAGs include hyaluronic acid, heparin,heparin sulfate, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatansulfate, and keratin sulfate. Other GAGs also can be used for formingthe matrix described herein, and those skilled in the art will eitherknow or be able to ascertain other suitable GAGs using no more thanroutine experimentation. For a more detailed description ofmucopolysaccharides, see Aspinall, "Polysaccharides", Pergamon Press,Oxford (1970).

Collagen can be reacted with a GAG in aqueous acidic solutions,preferably in diluted acetic acid solutions. By adding the GAG dropwiseinto the aqueous collagen dispersion, coprecipitates of tangled collagenfibrils coated with GAG results. This tangled mass of fibers then can behomogenized to form a homogeneous dispersion of fine fibers and thenfiltered and dried.

Insolubility of the collagen-GAG products can be raised to the desireddegree by covalently cross-linking these materials, which also serves toraise the resistance to resorption of these materials. In general, anycovalent cross-linking method suitable for cross-linking collagen alsois suitable for cross-linking these composite materials, althoughcrosslinking by a dehydrothermal process is preferred.

When dry, the cross-linked particles are essentially spherical withdiameters of about 500 μm. Scanning electron microscopy shows pores ofabout 20 μm on the surface and 40 μm on the interior. The interior ismade up of both fibrous and sheet-like structures, providing surfacesfor cell attachment. The voids interconnect, providing access to thecells throughout the interior of the particle. The material appears tobe roughly 99.5% void volume, making the material very efficient interms of the potential cell mass that can be grown per gram ofmicrocarrier.

Another useful synthetic matrix is one formulated from biocompatible, invivo biodegradable synthetic polymer, such as those composed of glycolicacid, lactic acid and/or butyric acid, including copolymers andderivatives thereof. These polymers are well described in the art andare available commercially. For example, 50:50 mixtures of poly D,Llactide: glycolide are commercially available from Boehringer Ingelheim(e.g., RG503, RG506, RG502H and RG503H), and Birmingham Polymers (e.g.,Lactel). In addition, polymers comprising 80% polylactide/20% glycosideor poly 3-hydroxybutyric acid may be purchased from PolySciences, Inc.The polymer compositions generally are obtained in particulate form andthe osteogenic devices preferably fabricated under aqueous conditions incombination with an organic solvent (e.g., in an ethanol-trifluoroaceticacid solution, EtOH/TFA). In addition, one can alter the morphology ofthe particulate polymer compositions, for example to increase porosity,using any of a number of particular solvent treatments known in the art.

III. Fabrication of the Osteogenic Device

The naturally sourced and recombinant proteins as set forth above, aswell as other constructs, can be combined and dispersed in a suitablematrix preparation using any of the methods described herein and/or inU.S. Pat. No. 5,266,683, the disclosure of which is incorporated byreference hereinabove.

Typically, osteogenic protein is dissolved in a suitable solvent andcombined with the matrix. The components are allowed to associate.Typically, the combined material then is lyophilized, with the resultthat the osteogenic protein is disposed on, or adsorbed to the surfacesof the matrix. Useful solubilizing solvents include, without limitation,an ethanol/trifluoroacetic acid solution (e.g., 47.5% EtOH/0.01% TFA);an acetonitrile/TFA solution; ethanol; ethanol in water; or aqueousbuffers. Formulations in an acidic buffer can facilitate adsorption ofOP-1 onto the matrix surface. For the devices of the invention a usefulformulation protocol is incubation of matrix and osteogenic protein inan ethanol/TFA solution (e.g., 30-50% EtOH/0.01-0.1% TFA) for 24 hours,followed by lyophilization. This procedure is sufficient to adsorb orprecipitate 70-90% of the protein onto the matrix surface.

The quantity of osteogenic protein used will depend on the size ofreplacement device to be used and on the specific activity of theosteogenic protein. Greater amounts may be used for large implants.Clinical formulations for large bony defects currently compriseapproximately 2.5 mg osteogenic protein per g of collagen matrix.

1. Ethanol Triflouracetic Acid Lyophilization

In this procedure, osteogenic protein is solubilized in an ethanoltriflouracetic acid solution (47.5% EtOH/0.01% TFA) and added to thecarrier material. The flurry is mixed and then lyophilized. This methodcurrently is preferred.

2. Acetonitrile Trifluoracetic Acid Lyophilization

This is a variation of the above procedure, using an acetonitriletrifluoracetic (ACN/TFA) solution to solubilize the osteogenic proteinthat then is added to the carrier material. Samples are vigorouslyvortexed many times and then lyophilized.

3. Ethanol Precipitation

Matrix is added to osteogenic protein dissolved in guanidine-HCl.Samples are vortexed, incubated at a low temperature (e.g., 4° C.), andrevortexed. Cold absolute ethanol (5 volumes) is added to the mixturewhich is then stirred and incubated, preferably for 30 minutes at -20°C. After centrifugation (microfuge, high speed) the supernatant isdiscarded. The reconstituted matrix is washed twice with coldconcentrated ethanol in water (85% EtOH) and then lyophilized.

4. Urea Lyophilization

For those osteogenic proteins that are prepared in urea buffer, theprotein is mixed with the matrix material, vortexed many times, and thenlyophilized. The lyophilized material may be used "as is" for implants.

5. Buffer Lyophilization

Osteogenic protein preparations in a physiological buffer also can bevortexed with the matrix and lyophilized to produce osteogenicallyactive formulations.

Furthermore, the procedures described herein can be used to adsorb otheractive therapeutic drugs, hormones, and various bioactive species to thematrix for sustained release purposes. For example, in addition toosteogenic proteins, various growth factors, hormones, enzymes,therapeutic compositions, antibiotics, or other bioactive agents alsocan be adsorbed onto, or impregnated within, a substrate and releasedover time when implanted and the matrix slowly is absorbed. Thus,various known growth factors such as EGF, PDGF, IGF, FGF, TGF-∝, andTGF-β can be released in vivo. The matrix can also be used to releasechemotherapeutic agents, insulin, enzymes, enzyme inhibitors orchemotactic-chemoattractant factors.

IV. Sterilization

Following formulation, the resulting osteogenic devices are sterilizedby exposure to ionizing radiation, for example, by exposure to anelection beam or to gamma irradiation. Because high energy oxygen ionsand radicals may be generated from oxygen atoms associated with thedevice, residual air should be removed from the device prior toirradiation.

Any form of packaging may be used to hold the osteogenic device of theinvention, provided that the packaging is compatible with thesterilization process and the maintenance of sterility under storageconditions. Stoppered vials currently are the preferred packagingagents.

Sterilization is performed routinely after the device has been sealed ina vial. In the currently preferred approach, the air is evacuated fromthe vial by means of a vacuum prior to sealing. As an additionalprecaution, however, the evacuated vial can be filled with an inert gas,for example, helium, neon, argon, or nitrogen, before sealing.Alternatively, the vial can simply be purged with an inert gas prior tosealing.

The sealed devices subsequently are terminally sterilized by exposureto, for example, an electron beam or gamma irradiation. The devices canbe sterilized during manufacture, i.e., as an integral inline step inthe manufacturing process, or alternatively, the devices, oncefabricated, can be sent to commercial sterilization services, forexample, Isomedix (Northboro, Mass.) or RTI-Process Technology(Rockaway, N.J.) for irradiation.

The devices of the invention typically are irradiated with a dosagesufficient to provide a sterility assurance level of about 10⁻⁶, asrequired by the Federal Drug Administration for sterilizingbiomechanical devices. The actual dosages necessary for sterilizing aparticular device can readily be determined by consulting the referencetext "Association for the Advancement of Medical InstrumentationGuidelines," published in 1992. Guidelines are provided therein fordetermining the radiation dose necessary to achieve a given sterilityassurance level for a particular bioburden of the device. Dosages forsterilizing devices of the invention preferably are within the range ofabout 0.5 to about 4.0 megarads and most preferably are within the rangeof about 2.0 to about 3.5 megarads.

EXEMPLICATIONS

The means for making and using the osteogenic devices of the invention,as well as other material aspects concerning the nature and utility ofthese compositions, including how to make and how to use the subjectmatter claimed, will be further understood from the following whichconstitutes the best mode currently contemplated for practicing theinvention. It will be appreciated that the invention is not limited tosuch exemplary work or to the specific details set forth in theseexamples.

EXAMPLE 1

Bioactivity of a Terminally Sterilized Osteogenic Device Comprising OP-1and Bovine Collagen Carrier Material

Demineralized, guanidine-extracted bovine matrix was formulated withincreasing amounts of OP-1 using the ethanol/TFA protocol as describedherein. Briefly, OP-1 solubilized in ethanol/TFA was incubated withbovine collagen carrier matrix material for 3 hours. The mixture wasfrozen as an OP-1/matrix slurry and dried under a vacuum. Afterformulation, each device was transferred to glass vials and water wasadded to some of the devices. Then, the devices were purged with heliumfor 5 minutes prior to sealing. The vials were closed with rubberseptums and sealed. Samples were irradiated with 2.5 megarads of gammairradiation under one of the following conditions: dry on dry ice, weton dry ice, or dry at room temperature.

The resulting devices were evaluated for bone producing potential in therat subcutaneous assay. In vivo bone induction was assayed as describedby Sampath et al. (supra). Briefly, terminally sterilized devices wereimplanted subcutaneously into recipient rats under ether anesthesia.Male Long-Evans rats, aged 28-32 days, were used. A vertical incision (1cm) was made under sterile conditions in the skin over the thoracicregion, and a pocket prepared by blunt dissection. Approximately 25 mgof the test device was implanted deep into the pocket and the incisionclosed with a metallic skin clip. The day of implantation was designatedas day zero of the experiment. Implants were removed on day 12. Theheterotropic site permits study of bone induction without the possibleambiguities resulting from the use of orthotropic sites.

Bone inducing activity was determined biochemically by means of analkaline phosphatase assay and calcium content of the day 12 implant.Alkaline phosphatase activity was determined spectrophotometricallyafter homogenization of the implant. Implants showing no bonedevelopment by histology have little or no alkaline phosphatase activityunder these assay conditions. The assay is useful for quantification andobtaining an estimate of bone formation quickly after the implants areremoved from the rat. Calcium content, on the other hand, isproportional to the amount of bone formed in the implant. Bone formationtherefore is calculated by determining the calcium content of theimplant on day 12 in rats.

Successful implants are characterized in that they exhibit a controlledprogression through the stages of protein-induced endochondral bonedevelopment, including: (1) transient infiltration by polymorphonuclearleukocytes on day one; (2) mesenchymal cell migration and proliferationon days two and three; (3) chondrocyte appearance on days five and six;(4) cartilage matrix formation on day seven; (5) cartilage calcificationon day eight; (6) vascular invasion, appearance of osteoblasts, andformation of new bone on days nine and ten; (7) appearance ofosteoclasts, bone remodeling and dissolution of the implanted matrix ondays twelve to eighteen; and (8) hematopoietic bone marrowdifferentiation in the ossicles on day twenty-one.

Histological sectioning and staining was performed to determine theextent of osteogenesis in the implants. Implants were fixed in BouinsSolution, embedded in paraffin, and cut into 6-8 μm sections. Stainingwith toluidine blue or hemotoxylin/eosin demonstrates clearly theultimate development of endochondral bone. Twelve day implants areusually sufficient to determine whether the implants contain newlyinduced bone. The results of the assays are summarized in Tables 1through 4 below.

                  TABLE 1                                                         ______________________________________                                        Device formulated with no OP-1/25 mg collagen                                                       2.5 megarads irradiation                                    (helium purge)                                                                    No        dry/rm                                                         Irradiation temp. dry/dry ice wet/dry ice                                  ______________________________________                                        Ca.sup.2+                                                                             2.9 ± 1.9                                                                            2.5 ± 2.3                                                                            7.9 ± 9.7                                                                          8.9 ± 8.6 (3)                            μg/mg tissue (3) (4) (5)                                                   Histology 1.3 ± 1.1 0 (4) 0 (5) 0 (3)                                      % Bone (3)                                                                    Alk. Phos. 0.15 ± 0.08 0.16 ± 0.03 0.10 ± 0.05 0.05 ± .02                                           (3)                                         Units/mg/30 (3) (4) (5)                                                       min.                                                                          weight 96.1 ± 7.8 48.0 ± 21.5 65.2 ± 18.2 81.5 ± 21.2 (3)                                            mg (1/2   (3) (4) (5)                      implant)                                                                    ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Device formulated with 500 ng OP-1/25 mg                                        collagen matrix.                                                                                  2.5 megarads irradiation                                    (helium purge)                                                                    No        dry/rm                                                         irradiation temp. dry/dry ice wet/dry ice                                  ______________________________________                                        Ca.sup.2+                                                                             27.0 ± 4                                                                             8.5 ± 5.8                                                                            20.8 ± 10.8                                                                        24.7 ± 10.0 (5)                          μg/mg tissue  (5) (6)                                                      Histology 60 ± 8 (4) 30 ± 20 (5) 43 ± 29 (6) 50 ± 28 (5)                                             % Bone                                     Alk. Phos. 1.02 ± 0.28 1.00 ± 0.47 1.15 ± 0.29 0.93 ± .21                                           (5)                                         Units/mg/30 (4) (5) (6)                                                       min.                                                                          weight 95.9 ± 11.6 80.2 ± 17.5 95.3 ± 22.0 90.4 ± 29.5 (5)                                           mg (1/2   (4) (5) (6)                      implant)                                                                    ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Device formulated with 1000 ng OP-1/25 mg                                       collagen matrix.                                                                                   2.5 megarads irradiation                                   (helium purge)                                                                     No        dry/rm                                                        irradiation temp. dry/dry ice wet/dry ice                                  ______________________________________                                        Ca.sup.2+                                                                              37.0 ± 7.8                                                                           19.9 ± 3.6                                                                           25.3 ± 10.5                                                                        29.6 ± 6.4                              μg/mg tissue (5) (5) (5) (50)                                              Histology 64 ± 11 (5) 52 ± 27 (5) 47 ± 33 (5) 46 ± 29 (5)                                             % Bone                                    Alk. Phos. 1.33 ± 0.37 1.33 ± 0.60 1.21 ± 0.63 1.40 ± 0.37                                            Units/mg/30 (5) (5) (5) (5)                                                   min.                                      Weight 149.1 ± 84.6 ± 16.7 97.9 ± 25.0 105.5 ±                    mg (1/2   37.8 (5) (5) (5) 14.2 (5)                                           implant)                                                                    ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Device formulated with 2500 ng OP-1/25 mg                                       collagen matrix.                                                                                   2.5 megarads irradiation                                 Irrad. No (helium purge)                                                    Conditions                                                                             irradiation                                                                             dry/rm temp                                                                             dry/dry ice                                                                           wet/dry ice                              ______________________________________                                        Ca.sup.2+                                                                              44.6 ± 14.9                                                                          29.0 ± 4.0                                                                           25.9 ± 11.7                                                                        46.4 ± 8.7                              μg/mg tissue (5) (5) (5) (5)                                               Histology 78 ± 13 (5) 74 ± 5 (5) 54 ± 21 (5) 78 ± 4 (5)                                               % Bone                                    Alk. Phos. 0.95 ± 0.19 1.32 ± 0.28 0.99 ± 0.21 1.43 ± 0.11                                            Units/mg/30 (5) (5) (5) (5)                                                   min.                                      Weight 205.4 ± 87.4 ± 25.5 133.6 ± 142.0 ±                        mg (1/2   47.5 (5) (5) 44.1 (5) 13.2 (5)                                      implant)                                                                    ______________________________________                                    

The data set forth in Tables 1 through 4 demonstrate that the osteogenicdevice can be sterilized by gamma irradiation as evidenced by theretention of biological activity of the OP-1 containing devices. Adecrease in osteogenic potential was seen in some groups. In general,the highest retention of activity was noted in the wet samplesirradiated on dry ice.

EXAMPLE 2

Alternative Procedures for Removing Air From Osteogenic Devices

Example 1 shows that under particular circumstances gamma irradiationmay reduce the bioactivity of the irradiated device. These results mayresult from incomplete removal of air from the devices prior toirradiation. In an attempt to minimize the reduction of bioactivitydifferent approaches were used to remove air from the devices prior toirradiation.

In a first method, the device containing vials were evacuated to lessthan 100 microns Hg using a lyophilizer and holding the vials underthese conditions for 5 minutes prior to sealing. In a second method, thedevice containing vials were evacuated to less than 100 microns Hg in alyophilizer, holding the samples for 5 minutes under these conditions,and then flushing the vials with helium for 2 minutes prior to sealing.The resulting samples were irradiated with 2.5 megarads of gammairradiation and subsequently analyzed for biological activity using therat subcutaneous assays as described in Example 1. The results aresummarized in Table 5.

Damage to the carrier matrix material was assessed by extracting matrixwith PBS and the absorbance of the resulting solution measured todetermine the amount of solubilized protein. Selected extracts wereanalyzed for amino acid composition which suggested that the solubilizedproteins were derived from collagen and represented between 1-2% of thestarting carrier matrix material.

                  TABLE 5                                                         ______________________________________                                        Effect of different approaches for removing air                                 from the devices prior to irradiation.                                                μg                                                                  OP-1/ Irradiation Conditions.sup.1                                               25 mg   No      Vacuum                                                                              Vacuum Helium Helium                                Assay carrier Irrad. RT Dry Ice RT Dry Ice                                  ______________________________________                                        Ca.sup.2+                                                                           0       0       0     0      0      0                                     μg/mg 0.5 23 ± 13 14 ± 12 5 ± 4 4 ± 5 13 ± 7                tissue 2.5 39 ± 11 25 ± 18 23 ± 7 30 ± 10 35 ± 9                                                        Histol- 0 0 0 0 0 0                  ogy 0.5 55 ± 33 29 ± 33 45 ± 26 36 ± 31 55 ± 22                % Bone 2.5 93 ± 5 63 ± 27 88 ± 4 83 ± 22 96 ± 50                                                        Weight 0 110 ± 150 ± 85                                                ± 24 105 ± 43 61 ± 34                                                 day 12  84 73                        Implant, 0.5 152 ± 121 ± 114 ± 46 99 ± 28 121 ± 32                                                      (mg)  23 46                           2.5 294 ± 193 ± 188 ± 53 171 ± 25 266 ± 31                      91 54                                                                     ______________________________________                                         .sup.1 All the devices except the control were sterilized with 2.5            megarads of gamma irradiation.                                           

The results indicate that devices irradiated on dry ice after purgingwith helium retained the highest levels of bioactivity. The results,however, indicate that devices irradiated under vacuum at roomtemperature also exhibit significant levels of bioactivity.

EXAMPLE 3

Reproducibility of Terminally Sterilized Devices

Five lots of devices were produced using the standard formulationprotocol. Briefly, OP-1 solubilized in 47.5% ethanol/0.01% TFA wasincubated with bovine collagen carrier matrix material for 3 hours. Themixture was frozen as an OP-1/matrix slurry and dried under a vacuum.After formulation, each device was transferred to glass serum vials, thevials sealed under vacuum and sterilized with 2.5-3.0 megarads of gammairradiation. All of the devices were formulated at 4° C. in glassformulation vessels. The results showing the extent of binding of OP-1to the matrices and the recovery of OP-1 from irradiated andnon-irradiated devices are set forth in Table 6.

The amount of OP-1 remaining in solution after 3 hours of incubation(prior to lyophilization of the device) was dependent on the ratio ofOP-1 to carrier matrix used in the formulation; the higher theconcentration of OP-1, the smaller the fraction of OP-1 bound to thematrix. When 2.5 mg OP-1 was formulated with one gram of matrix, theratio used to formulate a device for the clinical trials, between 53 and64% of the OP-1 was bound to the matrix after 3 hours incubation. Theamount of OP-1 recovered from the device before and after sterilizationis consistent from lot to lot of device, again showing thereproducibility of device production.

                  TABLE 6                                                         ______________________________________                                        Device formulated with OP-1.                                                                                       % OP-1                                        % OP-1 recovered                                                            mg OP-1/gm  bound at from device.sup.1                                     Device No.                                                                            matrix     Lot size 3 hr   non-irrad.                                                                           irrad.                              ______________________________________                                        P002-   3.12         5 grams                                                                              44     86     44                                    3, 4M15D1                                                                     P003- 1.25   1 gram 91 86                                                     1M15D1                                                                        P003- 2.50 1.5 grams 53 79 56                                                 1M15D2                                                                        P003- 5.00   1 gram 33 74                                                     1M15D3                                                                        Device Lot 2.50 5.9 grams 61 83 56                                            #1                                                                          ______________________________________                                         .sup.1 Samples of each of the devices were extracted with 8 M urea, 0.3%      Tween 80, 0.1 M NaCl and 50 mM Tris, pH 8.0 for 30 minutes at room            temperature. Aliquots of the extracts were analyzed for OP1 content using     HPLC.                                                                    

The data set forth in Examples 1-2 suggest that, under certainconditions irradiation can decrease the biological potency of device.These experiments were expanded to evaluate devices formulated at higherweight ratios of OP-1 to collagen matrix carrier material. The effectsof irradiation on the biological activity of the formulated devices wereevaluated in the rat subcutaneous assay. These devices were diluted withmatrix, specifically, irradiated device was diluted with irradiatedmatrix and a non-irradiated device was diluted with non-irradiatedmatrix, such that 3.12 mg of OP-1 and 25 mg of matrix were implantedinto the rats. The devices were diluted to avoid saturation of theassays. The results of each of the assays are set forth in Table 7. Nosignificant difference in the biological activity of irradiated andnon-irradiated device was observed.

                  TABLE 7                                                         ______________________________________                                        Biological Potency of Irradiated and Non-Irradiated Device.                               Irradiation                                                                             μg OP-1/                                                                          Biological Activity                              Lot     Dose      25 mg    Ca.sup.2+                                            Device (megarads) matrix (μg/mg tissue) Histology.sup.2                  ______________________________________                                        P002-   2.52-2/73 3.12     28.9 ± 4.3 (n = 6)                                                                    6/6                                       3M13D1                                                                        P002- none 3.12 35.2 ± 5.3 (n = 6) 6/6                                     3M13D1                                                                        P002- 2.80-2.99 3.12 28.6 ± 8.9 (n = 6) 6/6                                4, 5M13D4                                                                     P002- none 3.12 31.4 ± 14.1 (n = 6) 6/6                                    4, 5M13D4                                                                     P002- 2.66-2.94 3.12 26.5 ± 13.0 (n = 8) 8/8                               3, 4M15D1                                                                     P002- none 3.12 31.6 ± 15.4 (n = 8) 8/8                                    3, 4M15D1                                                                   ______________________________________                                         .sup.1 The irradiated OPdevices were diluted with irradiated matrix such      that 3.12 μg of OP1 and 25 mg matrix were implanted. Nonirradiated         device was diluted with nonirradiated matrix. These samples were evaluate     in the rat subcutaneous assay. The number of samples per assay are set        forth in parentheses.                                                         .sup.2 The histology results are presented as number of implants with         histological evidence of bone formation/total number of implants              evaluated.                                                               

EXAMPLE 4

Dilution of Formulations With Additional Carrier Material

In order to further assess any changes in the biological activity of thedevice accompanying irradiation, additional studies were conducted inwhich irradiated and non-irradiated devices were diluted with irradiatedmatrix and non-irradiated matrix to produce final formulationconcentrations of 3.1 μg OP-1/25 mg matrix and 1.5 μg OP-1/25 mg matrix.

Samples of the irradiated and non-irradiated devices were diluted withboth irradiated matrix and non-irradiated matrix. The samples wereevaluated using the rat subcutaneous assay and the results summarized inTable 8. The data again illustrate the biological potency of irradiateddevice.

An additional method for measuring changes accompanying irradiation ofthe device was to elute OP-1 from an irradiated device and to analyze itby HPLC, immunoblot and cellular assays. Results indicate thatirradiation of the device decreases by 30-50% the amount of OP-1 whichis eluted and detected by HPLC. Fractions from the HPLC were collectedand analyzed by immunoblot. The post-irradiated OP-1 elutes in the sameposition from the HPLC as the pre-irradiation sample, thus the decreasein recovery is not due to a change in the elution position of irradiatedOP-1. Samples of devices also were extracted with 2% SDS and theseextracts analyzed by immunoblot and in a cellular assay. Based onImmunoblot analysis of OP-1 eluted from a device with SDS, pre- andpost-irradiation, the protein pattern on the immunoblots lookssubstantially the same before and after irradiation, demonstrating thatthere is no significant physical change to the majority of the elutedOP-1 following terminal sterilization.

The OP-1 from two lots of collagen containing devices, lot numbersPOO2-3M13D1 (first device) and POO2-4,5M13D4 (second device) wasextracted with 2% SDS. These extracts were assayed in a rat osteoblastenriched cell assay, where the addition of OP-1 causes an increase inalkaline phosphatase activity (FIG. 1). In both cases, the activity ofextracts from irradiated device was approximately 70% of extracts fromnon-irradiated controls. In addition, the recovery of OP-1 fromirradiated device versus non-irradiated, as analyzed by HPLC, was 75%and 68% for device lot numbers POO2-3M13D1 and POO2-4,5M13D4,respectively.

                  TABLE 8                                                         ______________________________________                                        Biological Potency of Device Lot Number 1 Before                                and After irradiation.                                                                                           Biological                                 DEVICE DILUENT  Activity.sup.2                                                     non-            non-   μg OP-1/                                                                            Ca .sup.2+ μg/                        irrad. irrad. irrad. irrad. 25 mg matrix.sup.1 mg tissue                    ______________________________________                                        +               +             3.12     26.5 ± 13.0                           +   + 3.12 36.3 ± 17.3                                                      + +  3.12 33.4 ± 7.6                                                       +  + 3.12 31.6 ± 15.4                                                     +  +  1.56 30.7 ± 11.9                                                     +   + 1.56 39.5 ± 10.9                                                      + +  1.56 32.8 ± 13.0                                                      +  + 1.56 46.5 ± 8.6                                                    ______________________________________                                         .sup.1 Device was diluted with matrix to the final OP1 concentrations         outlined in this table. The device used for this experiment, Lot P0023,       4M15D1, was formulated with 3.12 mg OP1/gram matrix.                          .sup.2 Eight replicates of each combination were evaluated in the rat         subcutaneous assay. A portion of each implant was sent for histological       evaluation and the remainder was worked up for Ca.sup.2+ analysis. All of     the implants (64 of 64) showed histological evidence of bone formation.  

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

What is claimed is:
 1. A method of inducing endochondral bone formationor articular cartilage formation in a mammal, the method comprising thesteps of:(a) implanting at a preselected locus in the mammal, aterminally sterilized osteogenic device produced by the steps of:(i)providing an osteogenic device comprising in combinationa biocompatible,biodegradable insoluble carrier comprising a natural or syntheticpolymeric material and dimensioned to permit infiltration,proliferation, and differentiation of migratory progenitor cells whenimplanted into the mammal, and disposed therein, an isolated,biologically active osteogenic protein comprising a pair of disulfidebonded polypeptide chains which is competent to induce endochondral boneformation or articular cartilage formation when disposed in the carrierand implanted into the mammal, and (ii) exposing the osteogenic deviceof step (i) to ionizing radiation in an amount sufficient to achieve asterility assurance factor of 10⁻⁶ thereby to produce a terminallysterilized osteogenic device which is competent to induce endochondralbone formation or articular cartilage formation in the mammal; and (b)permitting the terminally sterilized osteogenic device to induceendochondral bone formation or articular cartilage formation at thepreselected locus.
 2. A method of producing a terminally sterilizedosteogenic device for implantation into a mammal, the method comprisingthe steps of:(a) providing an osteogenic device comprising incombination,a biocompatible, biodegradable insoluble carrier comprisinga natural or synthetic polymeric material and dimensioned to permitinfiltration, proliferation, differentiation of migratory progenitorcells when implanted into the mammal, and disposed therein, an isolated,biologically active osteogenic protein comprising a pair of disulfidebonded polypeptide chains which is competent to induce endochondral boneformation or articular cartilage formation when disposed in the carrierand implanted into the mammal; and (b) exposing the combination of step(a) to ionizing radiation in an amount sufficient to achieve a sterilityassurance factor of 10⁻⁶ thereby to produce a terminally sterilizedosteogenic device which is competent to induce endochondral boneformation or articular cartilage formation in the mammal.
 3. The methodof claim 1 or 2, wherein the osteogenic protein is OP-1, OP-2, BMP-2,BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, DPP, Vg1, or Vgr-1.
 4. Themethod of claim 1 or 2, wherein the osteogenic protein is OP-1.
 5. Themethod of claim 1 or 2, wherein the osteogenic protein is a member of asubclass of the superfamily of the TGF-β-like proteins.
 6. The method ofclaim 1 or 2, wherein the osteogenic protein is a recombinant osteogenicprotein.
 7. The method of claim 1 or 2, wherein the osteogenic proteinis a homodimer.
 8. The method of claim 1 or 2, wherein the syntheticpolymeric material comprises polylactic acid, polybutyric acid,polyglycolic acid, or a mixture thereof.
 9. The method of claim 1 or 2,wherein the natural polymeric material is hydroxyapatite, tricalciumphosphate, collagen, or a mixture thereof.
 10. The method of claim 1 or2, wherein the natural polymeric material is allogenic or xenogenicbone.
 11. The method of claim 1 or 2, wherein the insoluble carriercomprises porous particles.
 12. The method of claim 1 or 2, wherein theinsoluble carrier comprises packed particles.
 13. The method of claim 1or 2, wherein the insoluble carrier comprises particles having aparticle size within the range of about 70 to about 850 microns.
 14. Themethod of claim 13, wherein the insoluble carrier comprises particleshaving a particle size within the range of about 125 to about 450microns.
 15. The method of claim 1 or 2, wherein the carrier and theosteogenic protein are combined in a weight ratio ranging from about 1:1to about 250,000:1.
 16. The method of claim 15, wherein the carrier andthe osteogenic protein are combined in a weight ratio ranging from about40:1 to about 50,000:1.
 17. The method of claim 1 or 2, wherein theionizing radiation is gamma radiation.
 18. The method of claim 17,wherein the ionizing radiation is provided at a dose within the range ofabout 0.5 to about 4.0 megarads.
 19. The method of claim 18, wherein theionizing radiation is provided at a dose within the range of about 2.0to about 3.5 megarads.
 20. The method of claim 1 or 2, wherein theionizing radiation is an electron beam.
 21. The method of claim 20,wherein the ionizing radiation is provided at a dose within the range ofabout 0.5 to about 4.0 megarads.
 22. The method of claim 21, wherein theionizing radiation is provided at a dose within the range of about 2.0to about 3.5 megarads.
 23. The method of claim 1 or 2, wherein theterminally sterilized osteogenic device has at least about 20% of thebiological activity as the osteogenic device prior to sterilization. 24.The method of claim 1 or 2, wherein the terminally sterilized osteogenicdevice is competent to induce articular cartilage formation at anavascular locus in the mammal.